The enteric nervous system (ENS) is derived from cells that migrate from the neural crest and populate the anlage of the gut. Although the mechanisms that control the generation of specific cell types are largely unknown, we suggest that neural crest-derived cells acquire ENS specific phenotypic properties in the bowel as a consequence of integrating activities of gut-derived signals with intrinsic transcriptional activators. Despite the paucity of detailed information about the actions of transcriptional regulators in the ENS, we have cloned the basic helix-loop-helix transcription factor HAND2 (dHAND), whose neuronal expression is confined to enteric and sympathetic neural crest-derived neurons. HAND2 is a transcriptional regulator capable of coordinating expression of pan-neuronal characteristics with cell-type specific marker genes. The goal of the proposal is to establish the role that HAND2 gene products play in the differentiation of enteric neurons, and to identify genes interacting with HAND2 for the regulation of cell type-specific gene expression within the ENS. To ascribe a functional role for HAND2 gene products in development of enteric neurons we will first define the phenotypic properties in sub-classes of enteric neurons which express HAND2 and demonstrate the effects on development of blocking expression of HAND2 in enteric-derived neural precursors. Next, using both gain-of-function and loss-of-function paradigms, we will explore whether HAND2 supports neurogenesis in gut-derived precursors and/or couples aspects of neural specification with subtype-specific gene expression. This determination will allow us to distinguish a role in specification versus differentiation and provide a framework in which to assess likely co-activators with which HAND2 protein interacts. We will distinguish effects on neurogenesis from those on cell type-specific gene expression in transient transfection assays. Functional domains within HAND2 will be delineated using domain-mapping strategies in vitro and in ovo. We will ascertain if HAND2 functions in neurotransmitter/neuropeptide choice by investigating the ability of HAND2 to influence the transcription of regulatory molecules required for neurotransmitter biosynthesis. Delineating how phenotypic diversity is generated in the ENS will enhance our understanding of basic developmental mechanisms underlying neurogenesis, and identify the origins of clinical manifestations of problems with gut motility.